Non-heme diiron enzymes represent a diverse class of proteins that are responsible for a variety of biological functions by binding and activating dioxygen. Some of the members of this growing family of proteins include methane monooxygenase, ribonucleotide reductase, stearoylacyl carrier protein d9-desaturase, myo-inositol oxygenase, and ferritins. While the binuclear metal binding sites among most of the members of this family are composed of the same 4 Glu and 2 His ligand set, they perform dissimilar reactions. How these proteins are able to perform such diverse tasks using similar active sites remains a challenging question. The answer to this question will help us understand essential details of how the environment surrounding the active site acts to tune and modulate the reactivity of the binuclear metal binding sites. To this end, minimal model proteins that incorporate elements that are responsible for reactivity have generated significant insight into the relationship between the protein environment and the reactivity of diiron proteins. They possess the ability to house an active site within the core of a 4 helix bundle protein, yet are small enough that they are readily expressed, mutated, and studied using a number of structural and spectroscopic methods. Here, a novel artificial non-heme diiron protein that mimics the unusual protein AurF is described. AurF is a recently discovered N-hydrogenase involved in the biosynthesis of the polyketide antibiotic aureothin. When the metal binding site of AurF (4 Glu/3 His) was installed into the previously constructed artificial minimal diiron enzymes, it was found to also have N-hydrogenase activity. The goal of this project will be to understand how the environment surrounding the metal binding sites affects the N-hydrogenase activity of the novel DFscH3 model protein. Structural and spectroscopic measurements will be used to measure the changes in the metal ligand environment and its reactivity upon the creation of new constructs with mutations in the second/third ligand shell, metal binding ligand set, and the substrate access channel. The information gained from these studies will be correlated to how natural non-heme diiron enzymes use the protein environment to induce different reactivity from similarly built metal binding sites.

Public Health Relevance

Non-heme diiron proteins are essential enzymes that catalyze a variety of reactions through the activation of dioxygen and they are involved in a wide range of metabolic functions. Minimal model proteins that mimic the much larger and complex natural proteins can be extremely useful in deciphering the elements responsible for the enzyme reactivity. Here we seek to explore an artificial minimal model of the naturally occurring protein AurF, which is involved in the biosynthesis of the polyketide antibiotic aureothin.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04B-B (20))
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Fabian, Miles
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University of Pennsylvania
Schools of Medicine
United States
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Reig, Amanda J; Pires, Marcos M; Snyder, Rae Ana et al. (2012) Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins. Nat Chem 4:900-6